Association of myosteatosis with short-term outcomes in patients with acute-on-chronic liver failure

Sarcopenia (low muscle mass, i.e., quantity) is associated with poor clinical outcomes in patients with acute-on-chronic liver failure (ACLF). In this study, we aimed to illustrate the clinical prognostic value of myosteatosis (muscle fat infiltration) for short-term mortality in patients with ACLF. We retrospectively enrolled consecutive patients with ACLF between January 2019 and January 2022. Computed tomography-based body composition analysis was performed at the third lumbar vertebral level to determine skeletal muscle radiation attenuation. Fine and Gray’s competing risk regression model, with liver transplantation as a competing risk, was used to assess the factors associated with 90-day mortality. A total of 431 patients with ACLF were included. Myosteatosis and sarcopenia were observed in 261 (60.6%) and 87 (20.2%) patients, respectively. Competitive risk regression showed that age (HR 1.021, 95% CI 1.000–1.043, P = 0.042), APASL ACLF Research Consortium (AARC) score (HR 1.498, 95% CI 1.312–1.710, P < 0.001), and sarcopenia (HR 1.802, 95% CI 1.062–3.060, P = 0.029) were independently associated with increased 90-day mortality. Subgroup analysis of male patients with HBV-ACLF revealed that myosteatosis (HR 2.119, 95% CI 1.101–4.078, P = 0.025) was promising prognostic factors for 90-day mortality after being adjusted for ascites, acute kidney injury, AARC score, and sarcopenia. Myosteatosis is predictive of short-term outcomes in male patients with HBV-ACLF. Our results emphasise the importance of focusing on muscle fat infiltration in patients with HBV-ACLF. Further studies are warranted to investigate the underlying mechanisms and potential therapies for myosteatosis.


Body composition analysis
Abdominal noncontrast-enhanced CT was performed with the patient in the supine position using a 64-row CT scanner (GE LightSpeed).The technical parameters for the CT imaging were as follows: tube voltage, 120 kV; tube current, 380 mA; detector collimation, 0.625 mm; slice thickness, 5 mm; reconstruction thickness, 0.625 mm; and pitch, 5 mm.A single cross-sectional image at the L3 level was analysed by two hepatologists (NG and JZ) using the image analysis software SliceOmatic (V5.0;Tomovision, Magog, Canada).All skeletal muscle areas (SMA) at L3 were identified and quantified using a Hounsfield unit (HU) range of − 29 to 150.The HU thresholds were − 150 to − 50 for visceral adipose tissue area (VATA) and − 190 to − 30 for subcutaneous adipose tissue area (SATA).Cross-sections of the L3-SMA, VATA, and SATA were automatically calculated and normalised for height squared to obtain the skeletal muscle index at the third lumbar vertebra (L3-SMI), visceral adipose tissue index (VATI), and subcutaneous adipose tissue index (SATI) (cm 2 /m 2 ).Patients were classified as having sarcopenia using our previously established cut-off values: L3-SMI ≤ 40.2 cm 2 /m 2 in males and L3-SMI ≤ 31.6 cm 2 / m 2 in females 15 .Visceral adiposity was defined as VATA > 100 cm 216 .Sarcopenic obesity was defined as a combination of reduced muscle mass and increased BMI (> 25 kg/m 2 ) or increased VATA (> 100 cm 2 ) 14 .Myosteatosis was defined as SM-RA < 41 Hounsfield unit (HU) in patients with a body mass index (BMI) < 25 kg/m 2 and SM-RA < 33 HU in patients with a BMI ≥ 25 kg/m 217 .

Statistical analyses
Continuous variables are presented as mean ± standard deviation (SD) or median (interquartile range) for normally and non-normally distributed data.Categorical data are presented as numbers (percentages).Significant between-group differences were compared using independent sample t-tests or the Mann-Whitney U test.Significant differences between multiple groups were analysed using one-way analysis of variance or the Kruskal-Wallis test coupled with post hoc comparisons.Categorical data were compared using χ 2 tests.Univariate and multivariate logistic regression analyses were used to identify the risk factors for myosteatosis.The cumulative 90-day mortality according to the presence of myosteatosis were created in the total population as well as subgroup stratified by sex and etiology and compared with gray's test.Fine and Gray's competing risk regression model with liver transplantation as a competing risk factor was used to assess the risk factors associated with 90-day mortality.All the images were reviewed by two observers (NG and JZ) to assess inter-observer

Prognostic role of myosteatosis in male patients with HBV-ACLF
Female sex and alcoholic liver disease were independent risk factors for myosteatosis.As a continuous or categorical variable, SM-RA level and incidence rate of myosteatosis varied significantly among aetiologies and sexes (Supplementary Fig. 2).Therefore, incorporating different aetiologies and sex into the analysis may have confounding effects.In Asia, hepatitis B is the main cause of ACLF, and males account for over 80% of ACLF cases.Therefore, we investigated the 90-day prognostic value of myosteatosis in male patients with HBV-ACLF.

Discussion
Sarcopenia was significantly associated with the 90-day mortality in patients with all-cause ACLF and subgroup analysis of males with HBV-ACLF.Sarcopenia has become a popular topic in recent years, and its importance is being recognized by more and more disciplines.However, according to our diagnostic criteria for sarcopenia, its incidence rate was low, 20.2% in the total cohort, and only 14.4% in male patients with HBV-ACLF.Compared www.nature.com/scientificreports/with sarcopenia, there are fewer studies on myosteatosis, which is an emerging component of skeletal muscle abnormalities.To the best of our knowledge, this is the first study to evaluate the association between myosteatosis and short-term outcomes in patients with ACLF.Notably, the subgroup analysis of male patients with HBV-ACLF showed a strong association between myosteatosis and increased 90-day mortality rate.These results support the importance of focusing on muscle quality in patients with HBV-ACLF.Various diagnostic criteria can characterise myosteatosis, depending on reference populations and outcomes, without a clear consensus.Many studies have diagnosed myosteatosis based on a lower SM-RA measured in HU on CT, which represents an increased proportion of intramuscular adipose tissue and intramyocellular lipids.The widely used cutoff values of < 33 HU in patients with BMI ≥ 25 kg/m 2 and < 41 for BMI < 25 kg/m 2 were derived from a cancer population 17 .Additionally, muscle quality evaluation by CT at L3 using the intramuscular adipose tissue content (IMAC) parameter was initially first proposed to explore the association of IMAC with   19 .In summary, we adopted the most widely used method to diagnose myosteatosis.The mechanism contributing to excessive lipid accumulation within skeletal muscles is not completely understood, but may be related to metabolic abnormalities and mitochondrial dysfunction 3 .Excess lipids "overflow" from adipocytes and redistribute to other tissues, especially skeletal muscles 20 .In the early phase of lipid overload, oxidative muscles increase-oxidation capacity to inhibit excessive lipid deposition in muscle cells.Following excessive exposure to lipids, fatty acid oxidation decreases, and lipids are deposited due to mitochondrial dysfunction of the oxidative muscle.Body composition is characterised by sex-specific differences in muscle homeostasis and metabolism.We previously demonstrated that in healthy individuals, skeletal muscle mass and visceral adipose tissue in males are significantly higher than that in females, while subcutaneous adipose tissue in females is significantly higher than that in males 15,16 .In the present study, female patients had lower SM-RA levels and were more likely to develop myosteatosis.Differences in muscle types and sex differences in muscle metabolism are factors that must be considered when studying myosteatosis 21 .Liver disease may have different effects on skeletal muscle metabolism.In this study, we confirmed a strong association between alcoholic liver disease and myosteatosis.Ethanol can impair muscle protein homeostasis, promote fat accumulation, and increase the sensitivity of skeletal muscles to hyperammonaemia.Moreover, patients with non-alcoholic fatty experience increased fatty acid and insulin resistance, potentially inducing additional adverse effects, such as metabolic dysregulation 22 .Considering the significant differences between sexes and aetiologies, myosteatosis should be studied in sex-and aetiology-specific populations.
Muscle fat infiltration may not occur simultaneously with the loss of muscle mass.Whether Muscle fat infiltration is caused by the loss of muscle mass remains unclear, and the association between myosteatosis and sarcopenia remains controversial.Notably, our results showed that sarcopenia is closely related to myosteatosis.Similarly, a study conducted including 362 patients with chronic liver disease demonstrated that older age, female sex, presence of sarcopenia, and higher VSR levels were significant independent factors associated with myosteatosis 23 .In contrast, a retrospective analysis of 473 patients with decompensated cirrhosis revealed that advanced age, higher VSR, and higher VATI were independently associated with myosteatosis; however, there was no interaction between sarcopenia and myosteatosis 24 .This discrepancy may be attributed to the different thresholds and methods used to identify myosteatosis.We speculate that the excessive accumulation of lipids within skeletal muscles can lead to a decrease in muscle mass and dysfunction, and vice versa.The probable mechanisms are as follows: (1) myosteatosis may induce skeletal muscle mitochondrial dysfunction, insulin resistance, differentiation of muscle stem cells into adipocytes, and lipotoxicity, which inhibits protein synthesis and leads to decreased muscle mass and function.(2) sarcopenia exacerbates lipid accumulation in skeletal muscles due to small oxidised muscle fibres and the inability to effectively carry out fatty acid mitochondria β Oxidation 3,25 .
The association between myosteatosis and mortality has been evaluated for several medical conditions (Supplementary Table 2).Myosteatosis is negatively correlated with clinical prognosis, particularly when predicting short-term outcomes.Recently Czigany et al. 10 investigated the role of myosteatosis in predicting perioperative outcomes in deceased donor OLT.In the first 3 months after OLT, patients with myosteatosis had more severe surgical complications, increased need for intraoperative blood transfusions, increased rates of early allograft dysfunction, higher comprehensive complication index scores, longer ICU and hospital stays, and higher procedural costs.Another study by the same group investigated the effects of myosteatosis on long-term graft and patient survival after OLT and multivariable analysis revealed that myosteatosis was an independent predictor of adverse 5-year survival.However, the significant effect of myosteatosis was lost in univariate and multivariate analyses, from which patients who died within the first 90 days after OLT were excluded 8 .These findings indicate that myosteatosis may be a good predictor of the short-term prognosis of patients with end-stage liver disease.The additive effects of sarcopenia and myosteatosis on poor prognosis have been well studied.For example, concomitant sarcopenia and myosteatosis are associated with worse survival than the respective conditions alone 26 .In our study, myosteatosis plays an important prognostic role in male patients with HBV-ACLF.Notably, the 90-day cumulative mortality in patients with coexisting sarcopenia and myosteatosis was higher than that of myosteatosis alone.Therefore, for patients with myosteatosis, it is essential to provide enhanced care immediately when they are on the waiting list.
The cause of death for the patient with ACLF may be attributed to severe infections and multiple organ failures, including liver, kidneys, brain, respiratory and circulatory systems.The high mortality in ACLF patients with myosteatosis may be related to the high incidence of infections, impaired liver regeneration and systemic inflammatory response.Infection is the main complication of end-stage liver disease.A recent study demonstrated that the high risk of mortality associated with myosteatosis was associated with a higher frequency of sepsis-related deaths in patients with cirrhosis 27 .Excess adipose accumulation in skeletal muscles may affect muscle fibre orientation and is associated with decreased muscle strength, physical performance, and reduced resistance to stressors.Timely liver regeneration depends on adequate availability of energy and metabolites, and a decline in body reserves may reduce liver regeneration ability 28 .Additionally, myosteatosis is associated with increased inflammatory cytokine release and abnormal myokine and adipokine secretion, which may activate and recruit macrophages by binding to chemokine receptors or toll-like receptor 4, leading to an inflammatory cascade 25 .Ectopic fat can induce the formation of NLRP3 inflammasomes, thereby activating caspase1 and subsequently IL-1β and IL-18, and exacerbating systemic inflammation 29 .
Vol:.( 1234567890 Our study had several limitations.First, this was a retrospective, single-center study.Further multicenter prospective studies are imperative to confirm the generalizability and applicability of our findings.Second, we analysed only the prognostic role of myosteatosis at baseline.It is unclear whether ACLF is an aggravating factor of myosteatosis and whether the dynamic decline in muscle quality is more meaningful in predicting the prognosis.Third, although we used the most widely used method to diagnose myosteatosis, the suitability of these cutoff points for our population remains to be verified.Lastly, we hypothesized that inflammation, and impaired liver regeneration are the reasons for the poor prognosis of myosteatosis; however, comprehensive inflammatory cytokines and liver regeneration indices were not available due to the retrospective design of our study.The pathophysiological mechanisms underlying the poor clinical outcomes of ACLF with myosteatosis require further study.Therefore, there is an urgent need to investigate the pathogenesis and treatment methods of myosteatosis, establish appropriate diagnostic methods, and verify our conclusions in a prospective multicentre cohort study.

Conclusion
Our findings indicate that myosteatosis is independently associated with advanced age, female sex, alcoholic liver disease, and sarcopenia and is considered an independent predictor of short-term outcomes in male patients with HBV-ACLF.Myosteatosis may serve not only as a prognostic factor but also as a therapeutic target.The study emphasises the importance of identifying individuals muscle fat infiltration among those with normal muscle mass in clinical settings and may lay a foundation for clinician to improve patient care and plan the optimal treatment.Further studies are warranted to investigate the pathophysiology and therapeutic strategies for myosteatosis.

Figure 1 .
Figure 1.Flow diagram for enrolment in the study.

Table 1 .
Baseline characteristics of total cohort and classified by myosteatosis in patients with ACLF.Continuous variables are shown as mean ± standard deviation or median (interquartile range) for normally and non-normally distributed continuous variables, respectively.Categorical variables are presented as numbers (percentages).a Independent sample t-test, b Mann-Whitney U test, or c χ 2 test was used to compare two groups of continuous or categorical variables.Vol.:(0123456789) Scientific Reports | (2024) 14:13609 | https://doi.org/10.1038/s41598-024-64420-xwww.nature.com/scientificreports/

Table 2 .
Factors associated with myosteatosis according to univariate and multivariate logistic analysis in patients with ACLF.Age, sex, aetiology, liver cirrhosis, sarcopenia, VSR, and sarcopenic obesity were included in the multivariate logistic analysis.

Table 3 .
Univariate and multivariate competing risk regression for predicting 90-day mortality in the total cohort.Age, ascites, AKI, infection, AARC score, sarcopenia, myosteatosis, and sarcopenic obesity were included in the multivariate analysis.

Table 4 .
Univariate and multivariate competing risk regression for predicting 90-day mortality in male patients with HBV-ACLF.Ascites, AKI, AARC score, sarcopenia, and myosteatosis were concluded in the multivariate analysis.